Marchantiales-derived unsaturated fatty acid synthetase genes and use of the same

ABSTRACT

A Δ5 fatty acid desaturase gene, a Δ6 fatty acid desaturase gene, and a Δ6 fatty-acid-chain elongase gene are isolated from a single species of Marchantiales. By introducing these genes into higher plants, transformed plants which can produce arachidonic acid and eicosapentaenoic acid (EPA) are obtained.

TECHNICAL FIELD

The present invention relates to Marchantiales (Marchantiapolymorpha)-derived unsaturated fatty acid synthetase genes, i.e. genesof Δ5 fatty acid desaturase, Δ6 fatty acid desaturase, and Δ6fatty-acid-chain elongase, and use thereof.

BACKGROUND ART

Polyunsaturated fatty acids (PUFAs) such as arachidonic acid,eicosapentaenoic acid (hereinafter referred to as “EPA” as appropriate)are contained in lipids of the cell membrane of humans, notably in thenervous system. These polyunsaturated fatty acids act as a precursor ofa bioactive substance such as prostaglandin and leukotriene, and arevery important pharmacological substances. In recent years, health foodscontaining arachidonic acid and EPA have been commercially available. Inaddition, fatty acids, which are used as a source material of detergentsand biodegradable plastics, have captured the spotlight as materialsubstances.

Polyunsaturated fatty acids are currently produced by extraction fromcultured microorganisms or fish oil. This raises problems of highproduction cost, increased energy consumption and waste, and limitedfish resources particularly in methods using fish oil.

The biosynthesis of arachidonic acid and EPA is believed to occur in aseries of reactions involving Δ6 desaturation, chain elongation, and Δ5desaturation, with linoleic acid and α-linolenic acid being startingmaterials of the reactions yielding the arachidonic acid andeicosapentaenoic acid, respectively. These reactions are catalyzed by aΔ6 fatty acid desaturase (“Δ6 desaturase” hereinafter), Δ6fatty-acid-chain elongase (“Δ6 chain elongase” hereinafter), and Δ5fatty acid desaturase (“Δ5 desaturase” hereinafter), respectively.

A gene for the Δ6 desaturase is cloned from several plant species. Forexample, the gene has been cloned from Phaeodactylum tricornutum,Physcomitrella patens, ceratodon purpureous, borage, lithospermumerythrorhizon, primrose, and anemone. Apart from plants, the Δ6desaturase gene has also been cloned from filamentous fungi, nematodes,cyanobacteria, rats, and humans (see Non-Patent Publication 1: Eur. J.Biochem. 269, p4105, 2002; Non-Patent Publication 2: Plant J. 15, p39,1998; Non-Patent Publication 3: Eur. J. Biochem., 267. p3801, 2000;Non-Patent Publication 4: Proc. Natl. Acad. Sci. USA 94, p4211, 1997;Non-Patent Publication 5: Lipids 37, 417, 2002; Non-Patent Publication6: FEBS Lett. 542, p100, 2003; Non-Patent Publication 7: Whitney et al.,Planta Epub 2003; Non-Patent Publication 8: Lipids 34, p649, 1999;Non-Patent Publication 9: Gene, 238, p445 1999; Non-Patent Publication10: Biochem J. 330, p611 1998; Non-Patent Publication 11: Plant Mol.Biol., 22, p293 1993; Non-Patent Publication 12: Biochem. Biophys. res.Commun. 255, p575, 1999; and Non-Patent Publication 13: J. Biol. Chem.274, p471, 1999). All of these Δ6 desaturates cloned from theseorganisms, except that obtained from cyanobacteria, have a cytochrome b5domain at their N-terminus.

A gene of the Δ6 chain elongase was originally cloned from filamentousfungi and nematodes (see Non-Patent Publication 14: Proc. Natl. Acad.Sci. USA 97, p8284, 2000; and Non-Patent Publication 15: Proc. Natl.Acad. Sci. USA 97, p64-21, 2000). In plants, the gene has been clonedonly from Physcomitrella patens (see Non-Patent Publication 16: Plant J.31, p255, 2002).

In yeasts (Saccharomyces cerevisiae), there exist ELO2 protein and ELO3protein, which are involved in the synthesis of a long-chain saturatedacyl-chain of sphingolipids (see Non-Patent Publication 17: J. Biol.Chem., 272, p17376, 1997). The Δ6 chain elongase has an amino acidsequence homologous to the ELO2 protein and ELO3 protein. On the otherhand, in plants, there exists β-ketoacyl-CoA synthase (KCS), which isanother type of fatty-acid-chain elongase. This enzyme catalyzes theelongation of long-chain saturated and monounsaturated fatty acids (seeNon-Patent Publication 15 and Non-Patent Publication 18: Plant Cell 7,p309, 1995). However, the KCS gene is not evolutionary related to the Δ6chain elongase gene, or yeast ELO2 and ELO3 genes (see Non-PatentPublications 15 and 16).

A gene of the Δ5 desaturase was originally cloned from filamentous fungi(Non-Patent Publication 19: J. Biol. Chem. 273, p29360, 1998; andNon-Patent Publication 20: J. Biol. Chem. 273, p19055). The Δ5desaturase has a cytochrome b5 domain at the N-terminus as does the Δ6desaturase. The Δ5 desaturase gene has also been cloned fromPhaeodactylum tricornutum, nematodes, rats, humans, Physcomitrellapatens, and others (see Non-Patent Publication 1; Non-Patent Publication21: FEBS Lett. 439, p215, 1998; Non-Patent Publication 22: Arch.Biochem. Biophys. 391, p8, 2001; Non-Patent Publication 23: J. Biol.Chem. 274, p37335, 1999; and Non-Patent Publication 24: J. Biol. Chem.278, 35115, 2003).

Terrestrial plants are classified into bryophytes (Bryophyta),pteridophytes, gymnosperms, and angiosperms. Among these groups ofterrestrial plants, bryophytes are known to have branched off first, andthey are classified into three groups: Mosses (class Bryosida),Liverworts (class Hepaticopsida), and Hornwortz. Marchantia polymorphais taxonomically closest to Physcomitrella patens among the foregoingorganisms, but the latter belongs to class Bryosida while the formerbelongs to subclass Marchantiidae of class Hepaticopsida. It is certainthat the foregoing three groups were branched off at least about 430million years ago. Therefore, contrary to their common name “moss,” thedifference between Physcomitrella patens and Marchantia polymorpha isevolutionary far too great to be called as a difference, as comparedwith the difference, for example, between Arabidopsis thaliana and rice,which are believed to have branched off 200 million years ago (seeNon-Patent Publication 25: 110361514143968_(—)0.html).

As a Marchantia polymorpha-derived polyunsaturated fatty acid synthetasegene, KCS-like MpFAE2 and MpFAE3 chain elongase genes have been obtained(see Non-Patent Publication 26: Biosci. Biotechnol. Biochem. 67, p605,2003; and Non-Patent Publication 27: Biosci. Biotechnol. Biochem. 67,p1667, 2003). However, MpFAE2 and MpFAE3 are not Δ6 chain elongasegenes.

As described earlier, many polyunsaturated fatty acid biosynthetic genesare cloned from various species of organisms. However, there is only afew reports in which polyunsaturated fatty acids having 20 or morecarbon atoms with a degree of unsaturation 4 or greater, such asarachidonic acid and EPA, were produced in plants. As an example, it hasbeen reported that Phaeodactylum tricornutum-derived Δ6 desaturase andΔ5 desaturase, and a Physcomitrella patens-derived Δ6 chain elongasewere expressed in Linum usitatissimum to produce arachidonic acid andEPA. However, this is not described in detail (see Non-PatentPublication 24).

As described earlier, polyunsaturated fatty acids, such as arachidonicacid and EPA, are produced by extraction from cultured microorganisms orfish oil. This raises problems of high production cost, increased energyconsumption and waste, and limited fish resources. Polyunsaturated fattyacids such as arachidonic acid and EPA have a plurality of double bondsin the molecule. This unique characteristic enables these fatty acids tobe used in various industrial products (e.g. films, biodegradableplastics, functional fabrics, lubricating oil, and material substancefor detergents). By producing such polyunsaturated fatty acids intransgenic plants, it will be possible to reduce production cost andrealize a more environmentally friendly production process. Once thepolyunsaturated fatty acids are mass-produced with oil plants by geneticrecombinant techniques, it will be possible to advantageously use suchoil plants as inexpensive source materials for many different purposes.

However, in the expression of foreign genes in plants, it is difficultto predict how well the genes will function in the plants because thegene expression involves transcription, translation, and modifications.Further, in the expression of more than one foreign gene, it isenvisaged that the expressed genes will function more desirably whenthey come from a single species of plant, as opposed to different plantspecies as in the case of Non-Patent Publication 24. Further, Marchantiapolymorpha, which belongs to phylum Bryophyta—the first terrestrialplants—has been receiving attention as a model of higher plants, andtheir genes are expected to function well in other plants. Therefore,once Marchantia polymorpha-derived polyunsaturated fatty acid synthetasegenes, i.e. Δ5 desaturase gene, Δ6 desaturase gene, and Δ6 chainelongase gene are obtained, it will be possible to efficientlyaccumulate arachidonic acid and EPA in plants by introducing these genesinto plants.

The Δ5 desaturase gene, Δ6 desaturase gene, and Δ6 chain elongase genehave been cloned from Physcomitrella patens, which also belong to phylumbryophyta as does Marchantia polymorpha. However, since Marchantiapolymorpha and Physcomitrella patens are evolutionary very distantspecies, it is not easy to obtain Marchantia polymorpha genes usingPhyscomitrella patens genes with the current level of technology.

DISCLOSURE OF INVENTION

The present invention was made in view of the foregoing problems, and anobject of the invention is to provide Marchantiales (Marchantiapolymorpha)-derived unsaturated fatty acid synthetase genes,specifically, Δ5 desaturase gene, Δ6 desaturase gene, and Δ6 chainelongase gene, that can produce arachidonic acid or EPA in higherplants. The invention also provides a method of use of such genes.

In accomplishing the present invention, the inventors of the inventionidentified genes that encode the Δ5 desaturase, Δ6 desaturase, and Δ6chain elongase, using cDNA clones derived from Marchantiales (Marchantiapolymorpha), and successfully transferred and expressed these genes inmethylotrophic yeasts (Pichia pastoris). As a result, the inventors havefound that proteins expressed by these genes had enzyme activities ofthe Δ5 desaturase, Δ6 desaturase, and Δ6 chain elongase, respectively.Specifically, the present invention includes:

(1) A Marchantiales-derived gene that hybridizes under stringentconditions with all of or part of a DNA nucleotide sequence, or itscomplementary sequence, of SEQ ID NO: 1, and encodes a protein having aΔ6 fatty acid desaturating activity.

(2) A gene that encodes a Marchantiales-derived protein having a Δ6fatty acid desaturating activity, and that (a) consists of a nucleotidesequence of SEQ ID NO: 1, or (b) hybridizes under stringent conditionswith a DNA nucleotide sequence, or its complementary sequence, of SEQ IDNO: 1.

(3) A gene that encodes a Marchantiales-derived protein having a Δ6fatty acid desaturating activity, and that (a) consists of a nucleotidesequence of from the 253rd to 1698th nucleotides of SEQ ID NO: 1, or (b)hybridizes under stringent conditions with a DNA nucleotide sequence offrom the 253rd to 1698th nucleotides, or its complementary sequence, ofSEQ ID NO: 1.

(4) A gene that encodes a Marchantiales-derived protein having a Δ6fatty acid desaturating activity, and that (a) encodes a protein with anamino acid sequence of SEQ ID NO: 2, or (b) encodes a protein with anamino acid sequence that has been modified by substitution, deletion,insertion, and/or addition of one or more amino acids of SEQ ID NO: 2.

(5) A Marchantiales-derived gene that hybridizes under stringentconditions with all of or part of a DNA nucleotide sequence, or itscomplementary sequence, of SEQ ID NO: 3, and encodes a protein having aΔ6 chain elongating activity.

(6) A gene that encodes a Marchantiales-derived protein having a Δ6chain elongating activity, and that (a) consists of a nucleotidesequence of SEQ ID NO: 3, or (b) hybridizes under stringent conditionswith a DNA nucleotide sequence, or its complementary sequence, of SEQ IDNO: 3.

(7) A gene that encodes a Marchantiales-derived protein having a Δ6chain elongating activity, and that (a) consists of a nucleotidesequence of from the 194th to 1066th nucleotides of SEQ ID NO: 1, or (b)hybridizes under stringent conditions with a DNA nucleotide sequence offrom the 194th to 1066th nucleotides, or its complementary sequence, ofSEQ ID NO: 1.

(8) A gene that encodes a Marchantiales-derived protein having a Δ6chain elongating activity, and that (a) encodes a protein with an aminoacid sequence of SEQ ID NO: 4, or (b) encodes a protein with an aminoacid sequence that has been modified by substitution, deletion,insertion, and/or addition of one or more amino acids of SEQ ID NO: 4.

(9) A Marchantiales-derived gene that hybridizes under stringentconditions with all of or part of a DNA nucleotide sequence, or itscomplementary sequence, of SEQ ID NO: 5, and encodes a protein having aΔ5 fatty acid desaturating activity.

(10) A gene that encodes a Marchantiales-derived protein having a Δ5fatty acid desaturating activity, and that (a) consists of a nucleotidesequence of SEQ ID NO: 5, or (b) hybridizes under stringent conditionswith a DNA nucleotide sequence, or its complementary sequence, of SEQ IDNO: 5.

(11) A gene that encodes a Marchantiales-derived protein having a Δ5fatty acid desaturating activity, and that (a) consists of a nucleotidesequence of from the 375th to 1829th nucleotides of SEQ ID NO: 5, or (b)hybridizes under stringent conditions with a DNA nucleotide sequence offrom 375th to 1829th nucleotides, or its complementary sequence, of SEQID NO: 5.

(12) A gene that encodes a Marchantiales-derived protein having a Δ5fatty acid desaturating activity, and that (a) encodes a protein with anamino acid sequence of SEQ ID NO: 6, or (b) encodes a protein with anamino acid sequence that has been modified by substitution, deletion,insertion, and/or addition of one or more amino acids of SEQ ID NO: 6.

(13) A protein encoded by a gene of any one of genes (1) through (12).

(14) A protein (a) consisting of an amino acid sequence of SEQ ID NO: 2,or (b) consisting of an amino acid sequence that has been modified bysubstitution, deletion, insertion, and/or addition of one or more aminoacids of SEQ ID NO: 2, and having a Δ6 fatty acid desaturating activity.

(15) A protein (a) consisting of an amino acid sequence of SEQ ID NO: 4,or (b) consisting of an amino acid sequence that has been modified bysubstitution, deletion, insertion, and/or addition of one or more aminoacids of SEQ ID NO: 4, and having a Δ6 chain elongating activity.

(16) A protein (a) consisting of an amino acid sequence of SEQ ID NO: 6,or (b) consisting of an amino acid sequence that has been modified bysubstitution, deletion, insertion, and/or addition of one or more aminoacids of SEQ ID NO: 6, and having a Δ5 fatty acid desaturating activity.

(17) An antibody which recognizes a protein of any one of proteins (13)through (16).

(18) A recombinant expression vector which comprises a gene of any oneof genes (1) through (12).

(19) A transformant into which a gene of any one of genes (1) through(12) is introduced.

(20) A plant into which at least a gene of any one of genes (1) through(12) is expressibly introduced, its progeny or vegetatively propagatedplants having the same characteristics, or a tissue of the plant.

(21) A plant into which at least a gene of any one of genes (1) through(12) is expressibly introduced and whose fatty acid composition isthereby modified, its progeny or vegetatively propagated plants havingthe same characteristics, or a tissue of the plant.

(22) A reproductive material of a plant (20) or (21).

(23) A method of producing fatty acids, using a plant or a plant tissueof (21).

(24) A material substance which includes at least one compound selectedfrom the group consisting of: γ-linolenic acid; dihomo-γ-linolenic acid;arachidonic acid; stearidonic acid; eicosatetraenoic acid; andeicosapentaenoic acids, which are obtained by a method of (23).

(25) A method of modifying a fatty acid composition, using at least agene of any one of (1) through (12).

(26) A gene detecting instrument comprising as a probe at least aportion of a nucleotide sequence, or its complementary sequence, of agene of any one of (1) through (12).

(27) A screening method of a gene or substance that regulates a proteinof any one of (13) through (16), using a protein of any one of (13)through (16).

(28) A gene or substance obtained by a screening method of (27).

It is to be noted that, in the present invention, the nucleotides A, C,G, and T indicate adenine, cytosine, guanine, and thymine, respectively,unless otherwise specified.

Other objects, features, and advantages of the invention will be madeclear by the descriptions below. Benefits of the invention will also bemade clear by the description referring to the attached drawing.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is an explanatory drawing illustrating a procedure ofconstructing a construct in which expression cassettes of MpDES6 gene,MpELO1 gene, and MpDES5 gene used in Example 6 are joined to oneanother.

BEST MODE FOR CARRYING OUT THE INVENTION

The following will describe an embodiment of the present invention. Itis to be noted that the invention is not limited in any way by thefollowing description.

Specifically, the following describes, in order of appearance, syntheticpathways of arachidonic acid and eicosapentaenoic acid (EPA), genes andproteins according to the invention, methods of obtaining proteins andgenes of the invention, and methods of use (usefulness) of genes andproteins according to the invention.

(1) Synthetic Pathways of Arachidonic Acid and Eicosapentaenoic Acid(EPA)

The biosynthesis of arachidonic acid and eicosapentaenoic acid (EPA) isbelieved to occur in a series of reactions involving Δ6 desaturation, Δ6chain elongation, and Δ5 desaturation, with linoleic acid andα-linolenic acid being starting materials of the reactions yielding thearachidonic acid and eicosapentaenoic acid, respectively. Thesereactions are catalyzed by Δ6 desaturase, Δ6 chain elongase, and Δ5desaturase, and are called an n-6 pathway (arachidonic acid syntheticpathway), or an n-3 pathway (EPA synthetic pathway).

Previous reports suggest that the Δ6 desaturase, Δ6 chain elongase, andΔ5 desaturase are involved in both n-6 pathway and n-3 pathway.Specifically, the Δ6 desaturase in the n-6 pathway converts linoleicacid (18:2D^(9,12), containing 18 carbon atoms, and two double bonds atpositions 9 and 12 (the same notation will be used below)) intoγ-linolenic acid (GLA; 18:3D^(6,9,12)). In the n-3 pathway, the Δ6desaturase converts α-linolenic acid (ALA; 18:3D^(9,12,15)) intostearidonic acid (STA; 18:4D^(6,9,12,15)). The Δ6 chain elongase in then-6 pathway converts GLA into dihomo-γ-linolenic acid (DGLA;20:3Δ^(8,11,14)), and in the n-3 pathway converts STA intoeicosatetraenoic acid (ETA; 20:4Δ^(5,8,11,14,17)). The Δ5 desaturase inthe n-6 pathway converts DGLA into arachidonic acid (20:4Δ^(5,8,11,14)),and in the n-3 pathway converts ETA into eicosapentaenoic acid (EPA;20:5Δ^(5,8,11,14,17))

(2) Genes According to the Present Invention

[Δ6 Desaturase Gene According to the Invention]

A Δ6 desaturase gene according to the present invention is aMarchantiales-derived gene that encodes a protein having a Δ6 fatty aciddesaturating activity. Specifically, the gene satisfies the followingconditions:

1. A gene with the nucleotide sequence of SEQ ID NO: 1;

2. A gene that hybridizes under stringent conditions with DNA of anucleotide sequence, or its complementary sequence, of SEQ ID NO: 1;

3. A gene that hybridizes under stringent conditions with part of DNA ofa nucleotide sequence, or its complementary sequence, of SEQ ID NO: 1;

4. A gene with a nucleotide sequence of from the 253rd to 1698thnucleotides of a nucleotide sequence of SEQ ID NO: 1, wherein thisportion of the nucleotide sequence is translated into a protein with anamino acid sequence of SEQ ID NO: 2;

5. A gene that hybridizes under stringent conditions with a nucleotidesequence of from the 253rd to 1698th nucleotides of a nucleotidesequence, or its complementary sequence, of SEQ ID NO: 1;

6. A gene that encodes a protein with an amino acid sequence of SEQ IDNO: 2; and

7. A gene that encodes a protein with an amino acid sequence that hasbeen modified by substitution, deletion, insertion, and/or addition ofone or more amino acids of the amino acid sequence of SEQ ID NO: 2.

[Δ6 Chain Elongase Gene According to the Present Invention]

A Δ6 chain elongase gene according to the present invention is aMarchantiales-derived gene that encodes a protein having a Δ6fatty-acid-chain elongating activity. Specifically, the gene satisfiesthe following conditions:

1. A gene with a nucleotide sequence of SEQ ID NO: 3;

2. A gene that hybridizes under stringent conditions with DNA of thenucleotide sequence, or its complementary sequence, of SEQ ID NO: 3;

3. A gene that hybridizes under stringent conditions with part of DNA ofa nucleotide sequence, or its complementary sequence, of SEQ ID NO: 3;

4. A gene with a nucleotide sequence of from the 194th to 1066thnucleotides of a nucleotide sequence of SEQ ID NO: 3, wherein thisportion of the nucleotide sequence is translated into a protein with anamino acid sequence of SEQ ID NO: 4;

5. A gene that hybridizes under stringent conditions with a nucleotidesequence of from the 194th to 1066th nucleotides of a nucleotidesequence, or its complementary sequence, of SEQ ID NO: 3;

6. A gene that encodes a protein with an amino acid sequence of SEQ IDNO: 4; and

7. A gene that encodes a protein with an amino acid sequence that hasbeen modified by substitution, deletion, insertion, and/or addition ofone or more amino acids of the amino acid sequence of SEQ ID NO: 4.

[Δ5 Desaturase Gene According to the Invention]

A Δ5 desaturase gene according to the present invention is aMarchantiales-derived gene that encodes a protein having a Δ5 fatty aciddesaturating activity. Specifically, the gene satisfies the followingconditions:

1. A gene with a nucleotide sequence of SEQ ID NO: 5;

2. A gene that hybridizes under stringent conditions with DNA of anucleotide sequence, or its complementary sequence, of SEQ ID NO: 5;

3. A gene that hybridizes under stringent conditions with part of DNA ofa nucleotide sequence, or its complementary sequence, of SEQ ID NO: 5;

4. A gene with a nucleotide sequence of from the 375th to 1829thnucleotides of a nucleotide sequence of SEQ ID NO: 5, wherein thisportion of the nucleotide sequence is translated into a protein with anamino acid sequence of SEQ ID NO: 6;

5. A gene that hybridizes under stringent conditions with a nucleotidesequence of from the 375th to 1829th nucleotides, or its complementarysequence, of SEQ ID NO: 5;

6. A gene that encodes a protein with an amino acid sequence of SEQ IDNO: 6; and

7. A gene that encodes a protein with an amino acid sequence that hasbeen modified by substitution, deletion, insertion, and/or addition ofone or more amino acids of the amino acid sequence of SEQ ID NO: 6.

As used herein, “under stringent conditions” means that hybridizationtakes place only when there is at least 90% identity, preferably atleast 95% identity, and more preferably at least 97% identity.

Hybridization may be carried out by a conventional method, as describedin J. Sambrook et al. Molecular Cloning, A Laboratory Manual, 2nd Ed.,Cold Spring Harbor Laboratory (1989), for example. Generally, the levelof stringency increases with increase in temperature and/or decrease insalt concentration (more difficult to hybridize), and more homologousgenes are obtained. Hybridization conditions are not particularlylimited, and hybridization can be suitably carried out under variousconditions known in the art. For example, hybridization can be carriedout under the following conditions: 42° C., 6× SSPC, 50% formamide, 1%SDS, 100 μg/ml salmon sperm DNA, 5×Denhardt's solution (1×SSPE; 0.18 Msodium chloride, 10 mM sodium phosphate, pH 7.7, 1 mM EDTA).

As the term is used herein, “Marchantiales” is not just limited toMarchantia polymorpha, but includes all organisms that belong tosubclass Marchantiidae and order Marchantiales. Among such Marchantialesorganisms, the following species are known to contain super longpolyunsaturated fatty acids (Prog. Lipid Res. 32, p281, 1993): Monocleaforsteri (Monocleales), Corsinia coriandrina (Marchantiales), Oximitrapaleacea (Marchantiales), Ricciocarpos natans (Marchantiales), Riccahuebeneriana (Marchantiales), Ricca fluitans (Marchantiales), Riccaduplex (Marchantiales), Ricca canaliculata (Marchantiales), Riccabifurca (Marchantiales), Ricca ciliifera (Marchantiales), Ricca glauca(Marchantiales), Ricca sorocarpa (Marchantiales), Ricca warnstorfii(Marchantiales), Ricca michelii (Marchantiales), Ricca papillosa(Marchantiales), and Ricca zachariae (Marchantiales). With the currenttechniques, the Δ6 desaturase, Δ6 chain elongase, and Δ5 desaturasegenes can readily be obtained from these organisms. For example, genesof related species encoding enzymes that exhibit the same function areknown to cross-hybridize.

Genes according to the present invention are not limited todouble-stranded DNA, and may be the sense strand or anti-sense strand ofdouble-stranded DNA or RNA. The anti-sense strand may be used as a probeor anti-sense compound. For DNA, cDNA or genomic DNA obtained by cloningtechniques, chemical synthesis techniques, or a combination of thesedifferent techniques may be used. Further, genes according to thepresent invention may include a sequence of an untranslated region(UTR), or a vector sequence (including expression vector sequence).

(3) Proteins According to the Present Invention

[Δ6 Desaturase Protein According to the Present Invention]

A Δ6 desaturase protein according to the present invention is aMarchantiales-derived protein that has a Δ6 fatty acid desaturatingactivity. Specifically, the protein satisfies the following conditions:

1. A protein encoded by the Δ6 fatty acid desaturase gene of theinvention defined in section (2) above;

2. A protein with an amino acid sequence of SEQ ID NO: 2; and

3. A protein with an amino acid sequence that has been modified bysubstitution, deletion, insertion, and/or addition of one or more aminoacids of the amino acid sequence of SEQ ID NO: 2.

[Δ6 Chain Elongase Protein According to the Present Invention]

A Δ6 chain elongase according to the present invention is aMarchantiales-derived protein that has a Δ6 chain elongating activity.Specifically, the protein satisfies the following conditions:

1. A protein encoded by the Δ6 chain elongase gene of the inventiondefined in section (2) above;

2. A protein with an amino acid sequence of SEQ ID NO: 4; and

3. A protein with an amino acid sequence that has been modified bysubstitution, deletion, insertion, and/or addition of one or more aminoacids of the amino acid sequence of SEQ ID NO: 4.

[Δ5 Desaturase Protein According to the Present Invention]

A Δ5 desaturase protein according to the present invention is aMarchantiales-derived protein that has a Δ5 fatty acid desaturatingactivity. Specifically, the protein satisfies the following conditions:

1. A protein encoded by the Δ5 fatty acid desaturase gene of theinvention defined in section (2) above;

2. A protein with an amino acid sequence of SEQ ID NO: 6; and

3. A protein with an amino acid sequence that has been modified bysubstitution, deletion, insertion, and/or addition of one or more aminoacids of the amino acid sequence of SEQ ID NO: 6.

As the term is used herein, the “Δ6 fatty acid desaturating activity”means that the enzyme has substrate specificity to linoleic acid andα-linolenic acid, and converts these acids to γ-linolenic acid andstearidonic acid, respectively. As the term is used herein, “Δ6 chainelongating activity” means that the enzyme has substrate specificity toγ-linolenic acid and stearidonic acid, and converts these acids todihomo-γ-linolenic acid and eicosatetraenoic acid, respectively.Further, as the term is used herein, the “Δ5 fatty acid desaturatingactivity” means that the enzyme has substrate specificity todihomo-γ-linolenic acid and eicosatetraenoic acid, and converts theseacids to arachidonic acid and eicosapentaenoic acid (EPA), respectively.

As used herein, “substitution, deletion, insertion, and/or addition ofone or more amino acids” means substitution, deletion, insertion, and/oraddition of preferably no more than 10, more preferably no more than 7,and further preferably no more than 5 amino acids, as enabled by amutant protein producing method known in the art, for example, such as asite-directed mutagenesis inducing method. Such mutant proteins are notlimited to those intentionally mutated by a known mutant proteinproducing method, but also include those prepared by isolating andpurifying mutant proteins that exist in nature.

Proteins according to the present invention are not particularly limitedas long as they are polypeptides consisting of amino acids formingpeptide bonds. For example, the proteins may be conjugated proteins withan additional non-peptide structure. Non-limiting examples of such anon-peptide structure include a sugar chain and isoprenoid group.

Further, proteins according to the present invention may includeadditional peptides. Examples of such additional peptides includevarious epitopes such as His, Myc, and Flag tagged to proteins of thepresent invention.

Further, proteins according to the present invention may be obtained byintroducing genes according to the present invention (genes encodingproteins of the present invention) into a host cell and expressing thegenes therein. Alternatively, the proteins may be obtained by isolatingand purifying from cells or tissues. Further, depending on theexpression conditions in the host cell, proteins according to thepresent invention may be fusion proteins with other proteins. Further,proteins according to the present invention may be chemicallysynthesized.

(4) Method of Obtaining Proteins and Genes According to the PresentInvention

A method of obtaining (producing) proteins and genes according to thepresent invention is not particularly limited. The following describessome representative methods.

[Method of Obtaining a Protein]

As mentioned above, a method of obtaining (producing) proteins of thepresent invention is not particularly limited. In one method, proteinsaccording to the present invention are simply purified from cells ortissues expressing the proteins. A purification method is notparticularly limited either. For example, a cell extract prepared fromcells or tissues by a known method is purified by a known method, usinga column for example.

Further, proteins according to the present invention may be obtained bya method employing a genetic engineering technique. In this case, forexample, a vector that has incorporated genes of the invention isexpressibly introduced into a host cell by a known method, and theproteins obtained by translating the genes in the cell are purified.

An expression vector used to introduce foreign genes into a host issuitably selected from a group of expression vectors that haveincorporated different types of promoters that become functional in thehost and express the genes. A host is also suitably selected fromdifferent types of hosts. For the purification of the proteins,different methods are used depending on the type of host or propertiesof the proteins used. For example, required proteins can be purifiedrelatively easily with use of a tag.

A method of preparing a mutant protein is not particularly limitedeither. For example, a mutant protein may be generated by introducing apoint mutation in the nucleotide sequence using conventional mutantprotein inducing methods, such as a site-directed mutagenesis (seeHashimoto-Gotoh, Gene 152, 271-275 (1995), for example) or PCR.Alternatively, a mutant protein may be generated by a method in whichmutant strains are produced by insertion of transposons. Further, acommercially available kit may be used to prepare mutant proteins.

A method of obtaining proteins of the present invention is not limitedto the foregoing methods, and a chemical synthesis method may be used.For example, proteins according to the present invention may besynthesized from genes of the present invention, using a cell-freeprotein synthesis solution.

[Method of Obtaining Genes]

A method of obtaining (producing) genes of the present invention is notparticularly limited either. For example, a method employingdifferential screening (subtraction cloning) may be used. In thismethod, direct hybridization is repeated in a test tube according to amethod known in the art, so as to concentrate required cDNA (genes ofthe invention).

The differential screening may be carried out in steps under theconditions normally employed. The resulting clones can then be analyzedin detail by creating a restriction enzyme map and by sequencing theclones. By the analysis, the presence or absence of DNA fragmentsincluding gene sequences of the present invention can be confirmed.

In another method of obtaining genes of the present invention, DNAfragments including genes of the present invention are isolated andcloned by a known method. For example, a probe is prepared thathybridizes specific to a portion of nucleotide sequences of genesaccording to the present invention, so as to screen the genomic DNAlibrary or cDNA library. The probe is not particularly limited and mayhave any sequence or length, as long as it can hybridize specific to atleast a portion of nucleotide sequences or their complementary sequencesof genes according to the present invention.

When a probe sequence is selected from a region well conserved amongdifferent species of Marchantiales, screening of genomic DNA or cDNA ofother species of Marchantiales using the probe enables isolation andcloning of genes that encode homologous or analogous moleculesfunctionally similar to proteins of the invention.

Further, in another method of obtaining genes of the present invention,amplifying means such as PCR may be used. For example, primers areprepared from the 5′ end and 3′ end of a cDNA sequence (or itscomplementary sequence) of genes according to the present invention.With these primers, PCR is carried out using the genomic DNA (or cDNA)as a template. The PCR amplifies a region of DNA flanked by the primers,thereby producing a large number of DNA fragments including genes of thepresent invention.

(5) Method of Use (Usefulness) of Genes and Proteins According to thePresent Invention

(5-1) Recombinant Expression Vector

A recombinant expression vector according to the present invention isnot particularly limited as long as it includes genes of the presentinvention described in section (2) above. For example, a recombinantexpression vector that has incorporated cDNA may be used. Therecombinant expression vector may be prepared using a plasmid, phage, orcosmid as non-limiting examples. Alternatively, the recombinantexpression vector may be prepared by a method known in the art.

The type of vector is not particularly limited as long as it isexpressed in a host cell. Specifically, such vectors are prepared byintroducing genes of the present invention in a plasmid along withpromoter sequences that have been suitably selected to ensure geneexpression. The promoter sequences depend on the type of host cell.

Various markers may be used to confirm whether genes of the presentinvention have been introduced in a host cell, or whether the genes havebeen successfully expressed in the host cell. For example, a marker (agene lacking in the host cell) is integrated with a carrier, such as aplasmid, together with genes of the present invention, and is introducedinto the host cell as an expression vector. Successful uptake of thegenes of the present invention may be confirmed by checking theexpression of the marker in the host cell that has incorporated theexpression vector. Alternatively, protein according to the presentinvention may be expressed in the form of fusion proteins in the hostcell. For example, proteins according to the present invention may beexpressed as fusion proteins with a green fluorescence protein (GFP)derived from Aequorea victoria. In this case, the GFP is used as amarker.

The host cell is not particularly limited, and various conventionallyavailable cells may be used. Non-limiting examples of such cellsinclude: bacteria such as Escherichia coli; yeasts such as Saccharomycescerevisiae or Schizosaccharomyces pombe; Caenorhabditis elegans; andoocytes of Xenopus laevis.

A method of introducing the expression vector into the host cell (methodof transformation) is not particularly limited and various conventionalmethods may be used, including an electroporation method, calciumphosphate method, liposome method, and DEAE dextran method, for example.Further, when proteins of the present invention are transferred andexpressed in insects, an expression system using baculovirus may beused.

(5-2) Transformants

Transformants according to the present invention are not particularlylimited as long as they incorporate genes of the present inventiondescribed in section (2) above. As the term is used herein,“transformants” means not only cells, tissues, or organs, but alsoliving organisms themselves.

A method of preparing (producing) transformants is not particularlylimited. For example, a host cell may be transformed by introducing arecombinant expression vector described above. The organisms to betransformed are not particularly limited, and may be microorganisms oranimals as exemplified above.

Further, transformants according to the present invention are preferablyplants into which genes of the present invention are expressiblyintroduced, or their progeny or vegetatively propagated plants havingthe same characteristics. Preferably, tissues of these plants also fallwithin the meaning of “transformants.” With these transformant plants,polyunsaturated fatty acids such as arachidonic acid and EPA can beproduced at low cost by an environmentally friendly process.

Further, as used herein, “expressibly introduce genes” means that genesare expressibly introduced into a target cell (host cell), using knowngenetic engineering (gene manipulation) techniques.

The recombinant expression vector used for the transformation of plantsis not particularly limited as long as it can express the inserted genesin the plant cell. Examples of such a vector include a vector with apromoter (for example, cauliflower mosaic virus 35S promoter) forconstitutively expressing genes in a plant cell, and a vector with apromoter that is inductively activated in response to external stimuli.Here, the plant cells include various types of plant cells, for example,such as cells in a suspension culture, protoplasts, slices of leaves,and calluses.

The recombinant expression vector may be introduced into a plant cell bya method known in the art, for example, such as a polyethylene glycolmethod, electroporation method, a method using Agrobacterium, and aparticle gun method. Reproduction of plants from the transformed cellsmay be carried out by a method known in the art.

For example, there have been established methods of obtainingtransformed tobacco, including: a method in which a transformedAgrobacterium is used to infect a tobacco leaf disc; a method in whichgenes are introduced into a protoplast using polyethylene glycol toreproduce plants; a method in which genes are introduced into aprotoplast by an electrical pulse to reproduce plants; and a method inwhich genes are directly introduced into a cell by a particle gun methodto reproduce plants. The present invention can suitably employ any ofthese methods.

Further, beside Arabidopsis thaliana, tobacco is also a model plant ofplant cultivation using genetic engineering techniques. Once atransformant tobacco with a high arachidonic acid or EPA content isobtained, it will be possible to obtain such transformants in all otherplants. In addition to transformant tobacco, the present invention alsoprovides transformant rice, as will be described later in Examples.Therefore, the invention is able to provide transformants in any type ofplant.

For example, there have been established methods of obtainingtransformed rice, including: a method in which genes are introduced intoa protoplast using polyethylene glycol to reproduce plants; a method inwhich genes are introduced into a protoplast by an electrical pulse toreproduce plants; and a method in which genes are directly introducedinto a cell by a particle gun method to reproduce plants. The presentinvention can suitably employ any of these methods.

With a transformant rice containing an increased level of arachidonicacid or EPA, intake of these and other polyunsaturated fatty acids ispossible only by eating the seeds (grains) of the transformant.Therefore, the transformant rice is highly valuable as a food crop, andis highly useful in food industries and agricultures. Further,arachidonic acid or EPA can also be produced in rice bran, chaff, ortiller, which are often wasted. By extracting fatty acids from theseparts of plant, they can be efficiently used as a source of health food.They can also be used as food of domestic animals.

Once a transformant plant is obtained that has incorporated genes of thepresent invention in its genome, progeny of the plant can be obtained byreproducing the plant either sexually or asexually. Further,reproductive materials, for example, such as seeds, fruits, cuttings,tuberous stems, tuberous roots, stumps, callus, and protoplasts may beobtained from the plant, or from its progeny or clones. From thesereproductive materials, the plant may be mass produced. The presentinvention therefore includes plants into which genes of the inventionare expressibly introduced, their progeny or vegetatively propagatedplants having the same characteristics, tissues of the plants, andreproductive materials of the plants. Further, the plants, their progenyor vegetatively propagated plants having the same characteristics, andtissues of the plants include plants that are reproduced vegetatively.Vegetative reproduction is also known as vegetative generation or clonalexpansion, and is commonly carried out by making a plant or herbaceouscutting. In a test tube, vegetative reproduction of plants may becarried out by redifferentiating plants from such organs as the leaf,stem, or root, or by using a callus. In some plant species, a uniquewinter bud may form at the tip of a branch, or a succulent axillary budmay be formed. In other cases, flowers may form a propagule, or a tubermay be formed.

Further, the present invention includes plants into which genes of thepresent invention are expressibly introduced, and whose fatty acidcomposition is modified by the expression of the genes. The inventionalso includes their progeny or vegetatively propagated plants having thesame characteristics, tissues of the plants, and reproductive materialsof the plants. As used herein, “modification of fatty acid composition”means altering of a fatty acid composition of plant by way oftransformation. For example, such a change can be induced by introducinggenes of the present invention to transform a plant which does notcontain arachidonic acid or EPA in its fatty acid composition. This willchange the fatty acid composition of the plant by the production ofarachidonic acid and EPA.

(5-3) Fatty Acid Producing Method

The present invention includes a producing method of fatty acids, usinga plant that has been transformed by genes of the present invention andthereby whose fatty acid composition has been modified, or using tissuesof the plant.

For example, food oil may be produced from a transformant according tothe present invention containing a high level of arachidonic acid orEPA. With the increased level of arachidonic acid or EPA, the value ofthe product food oil can be increased. Further, various parts of thetransformant plant, for example, such as seeds, fruits, cuttings,tuberous stems, and tuberous roots, can be used to supply arachidonicacid- or EPA-containing food with an increased value.

(5-4) Material Substance

The present invention includes a substance obtained by a fatty acidproducing method of the invention. Specifically, the invention includesa material substance which includes at least one compound selected fromthe group consisting of: γ-linolenic acid, dihomo-γ-linolenic acid,arachidonic acid, stearidonic acid, eicosatetraenoic acid, andeicosapentaenoic acid. As used herein, “material substance” means allsubstances usable as raw materials for various industrial purposes,including seeds, fruits, cuttings, tuberous stems, and tuberous roots,provided as food.

A material substance containing arachidonic acid or EPA may be used toprovide health food, film, biodegradable plastic, functional fiber,lubricant, and detergent, for example. The polyunsaturated fatty acidshave a unique property of containing more than one double bond withinthe molecule. Thus, by producing arachidonic acid or EPA in atransformant of the present invention for example, production cost canbe reduced. Further, the invention realizes an environmentally friendlyproducing process.

(5-5) Fatty Acid Composition Modifying Method

The present invention includes a method of modifying a fatty acidcomposition using genes of the present invention. For example, with atransformant that has incorporated genes of the present invention asabove, a fatty acid composition of the host cell can be modified. Atarget organism of fatty acid composition modification is notparticularly limited. Other than plants, any organism may be used,including animals, microorganisms, and yeasts.

(5-6) Gene Detecting Instrument

A gene detecting instrument according to the present invention includesat least a portion of a nucleotide sequence, or its complementarysequence, of a gene of the present invention as a probe. The genedetecting instrument of the invention can be used, under variousconditions, for the measurement or detection of expression pattern ofgenes according to the present invention.

An example of a gene detecting instrument according to the presentinvention is a DNA chip in which a probe that hybridizes specific togenes of the present invention is immobilized on a substrate (carrier).As used herein, the term “DNA chip” generally means a synthetic DNA chipusing a synthetic nucleotide as a probe, but the “DNA chip” also meansan adhesion DNA microarray that uses a PCR product, such as cDNA, as aprobe.

The probe sequence may be determined by a conventional method ofspecifying a characteristic sequence of cDNA sequences. For example, aSAGE (Serial Analysis of Gene Expression) method, as described inScience 276:1268, 1997; Cell 88: 243, 1997; Science 270: 484, 1995;Nature 389: 300, 1997; U.S. Pat. No. 5,695,937 may be used.

The DNA chip may be made by a conventional method. For example, when asynthetic oligonucleotide is used, it may be synthesized on a substrateby a combination of photolithography and solid phase DNA synthesistechnique. On the other hand, when the oligonucleotide is cDNA, it isstuck on a substrate using an array device.

Further, as in common DNA chips, the accuracy of gene detection can beimproved by placing a perfect-match probe (oligonucleotide) with amismatch probe that has been prepared by substituting a singlenucleotide of the perfect-match probe. Further, in order to detectdifferent genes simultaneously, a DNA chip may be prepared in whichdifferent types of oligonucleotides are immobilized on a singlesubstrate.

A gene detecting instrument of the present invention is not just limitedto the DNA chip as exemplified above, as long as it uses at least aportion of a nucleotide sequence, or its complementary sequence, of agene of the present invention as a probe.

(5-7) Antibody

An antibody according to the present invention is a polyclonal ormonoclonal antibody obtained by a method known in the art, usingproteins of the invention, or fragments of the proteins or peptides asan antigen. Examples of the conventional method include Harlow et al.;Antibodies: A laboratory manual (Cold Spring Harbor Laboratory, New York(1988), and Iwasaki et al.; Monoclonal antibody, hybridoma and ELIZA,Kodansha (1991). The antibody may be used in the detection and/ormeasurement of a protein according to the present invention.

(5-8) Screening Method

A screening method according to the present invention uses proteins ofthe present invention to screen for genes or substances that regulatethe proteins. A screening method of the invention is not particularlylimited, and a variety of conventional methods that find the presence orabsence of bonding or dissociation between substances may be used. Forexample, substances that facilitate the activities of proteins accordingto the present invention (Δ6 desaturase activity, Δ6 chain elongaseactivity, and/or Δ5 desaturase activity) may be screened.

The present invention also includes genes or proteins obtained by such ascreening method.

While the invention is susceptible to various modifications andalternative forms, a specific embodiment thereof will be described belowin more detail by way of Examples with reference to the attacheddrawings. It should be understood, however, that it is not intended tolimit the invention to the particular forms disclosed, but on thecontrary, the invention is to cover all modifications, equivalents, andalternatives falling within the scope of the invention as defined in theappended claims.

EXAMPLES

In the following Examples, experimental methods, unless otherwisespecified, are based on the method described in Molecular Cloning(Sambrook et. al. Cold Spring Harbour Laboratory Press, 1989).

Example 1 Isolation of Marchantia polymorpha-derived Δ6 Desaturase Gene

A comparison of amino acid sequences of cloned Δ6 desaturases hasconfirmed that the amino acid sequencesTrp-Trp-Lys-(Glu/Asp)-Lys-His-Asn (SEQ ID NO: 37) andTrp-Phe-Thr-Gly-Gly-Leu-Asn (SEQ ID NO: 38) were conserved. To isolate aMarchantia polymorpha-derived Δ6 desaturase gene, the followingdegenerate primers encoding the above amino acid sequences were used:

dΔ6DES-F: (SEQ ID NO: 7) 5′-TGGTGGAA(A/G)GA(A/G/T/C)AA(A/G)CA(T/C)AA-3′;and dΔ6DES-R: (SEQ ID NO: 8) 5′-(A/G)TTIA(A/G)ICCICCIGT(A/G)AACCA-3′.(“I” denotes inosine, and more than one nucleotide exist inparentheses.)

A thallus of E-line Marchantia polymorpha (see Transgenic Res. 9, p179,2000) was used as a sample. Isolation of poly(A)⁺ RNA from the thalluswas carried out in accordance with the method described in Biosci.Biotechnol. Biochem. 67, p605, 2003; Biosci. Biotechnol. Biochem. 67,p1667, 2003. 1.5 μl of isolated poly(A)⁺ RNA was reverse-transcribed tocDNA using a Ready-To-Go T-primed First Strand kit (Amersham). PCR wascarried out with about 10 ng of the cDNA as a template, using theforegoing primers (dΔ6DES-F and dΔ6DES-R) and 0.5 U of enzyme (Takara ExTaq, Takara), by a method recommended by the manufacturer. Using aGeneAmp PCR system 9700 (PE Applied Biosystems), the PCR was carried outwith 20 μl of a reaction solution under the following conditions: 94° C.for 2 minutes, followed by 35 cycles of reaction at 94° C. for 1 minute,45° C. for 1.5 minutes, and 72° C. for 2 minutes, and cooling down to 4°C.

The resulting PCR product was electrophoresed on a 1% (w/v) agarose gel,and amplified fragments having a size expected from a known amino acidsequence of a conventional Δ6 desaturase was collected from the gelusing a Prep-A Gene (Bio-rad). The collected fragments were ligated to apT7Blue Vector (Takara) and transformed into Escherichia coliElectro-max DH10B cells (Invitrogen, Carlsbad, Calif.).

Nucleotide sequences of all clones obtained by a BigDye Terminator CycleSequencing kit (Applied Biosystems) and an automated sequencer ABI PRISM377 (Applied Biosystems) were determined, and the clones were screenedfor a target cDNA sequence.

Further, to obtain a full-length cDNA sequence, 5′-RACE and 3′-RACE werecarried out by a method recommended by the manufacturer, using a 5′-RACESystem for Rapid Amplification of cDNA Ends Version 2.0 (Invitrogen), aReady-To-Go T-primed First Strand kit (Amersham), and the followingprimers:

MpDES6-02R: (SEQ ID NO: 9) 5′-AAGTTGCCTTCGATGTTTCTGG-3′; and MpDES6-01F:(SEQ ID NO: 10) 5′-GCTCGCCTGGAGCAAGGAAATC-3′.

As a result, one type of candidate homologue gene was isolated as anMpDES6 gene. The length of cDNA (not including a poly(A) portion) of theisolated MpDES6 gene was 2,522 bp, and an amino acid sequence encoded bythe MpDES6 gene was estimated to have 481 residues. The nucleotidesequence and amino acid sequence of the MpDES6 gene are represented bySEQ ID NO: 1 and SEQ ID NO: 2, respectively.

A comparison between the estimated amino acid sequence of MpDES6 cDNAand an amino acid sequence of Δ6 desaturase from Physcomitrella patensfound only 47.5% identity.

Example 2 Isolation of Marchantia polymorpha-Derived Δ6 Chain ElongaseGene

A comparison of amino acid sequences of cloned Δ6 chain elongases hasconfirmed that the amino acid sequences Val-Glu-Phe-Met-Asp-Thr-Val (SEQID NO: 39) and Lys-Tyr-Leu-Phe-Trp-Gly-Arg (SEQ ID NO: 40) wereconserved. To isolate a Marchantia polymorpha-derived Δ6 chain elongasegene, the following degenerate primers coding for the above amino acidsequences were used:

dΔ6ELO-F: (SEQ ID NO: 11) 5′-GTIGA(A/G)TT(T/C)ATGGA(T/C)ACIGT-3′; anddΔ6ELO-R: (SEQ ID NO: 12)5′-C(G/T)ICCCCA(A/G)AAIA(A/G)(A/G)TA(T/C)TT-3′.

PCR was carried out using the these primers (dΔ6ELO-F and dΔ6ELO-R), andthe resulting DNA fragments were subcloned. Nucleotide sequences of theclones were determined, and a full-length cDNA was obtained for clonesthat had a target cDNA sequence, using the following primers:

MpELO1-02R: (SEQ ID NO: 13) 5′-GCGAGCTTTCTCGTTCTTTCCC-3′; andMpELO1-01F: (SEQ ID NO: 14) 5′-TATGATTTTGAAGCGCAACACG-3′.Note that, the materials and methods for the experiment were the same asfor Example 1.

As a result, one type of candidate homologue gene was isolated as anMpELO1 gene. The length of cDNA (not including a poly(A) portion) of theisolated MpELO1 gene was 1,559 bp, and an amino acid sequence encoded bythe MpDES1 gene was estimated to have 290 residues. The nucleotidesequence and amino acid sequence of the MpDES1 gene were represented bySEQ ID NO: 3 and SEQ ID NO: 4, respectively.

A comparison between the estimated amino acid sequence of MpELO1 cDNAand an amino acid sequence of Δ6 chain elongase from Physcomitrellapatens found 62.7% identity.

Example 3 Isolation of Marchantia polymorpha-Derived Δ5 Desaturase Gene

The Δ5 desaturases of other species have a cytochrome b5 domain at theirN-terminus. From this, it was speculated that a Marchantiapolymorpha-derived Δ5 desaturase gene belongs to a cytochrome b5-domainfusion desaturase gene family, as does the Δ6 desaturase gene. However,in Phaeodactylum tricornutum and fungi, the homology between the Δ5desaturase and Δ6 desaturase is very poor at an amino acid level. Assuch, amino acid sequences of the Δ5 desaturase and Δ6 desaturase werecompared in a filamentous fungus (M. alpina). The comparison found localpresence a contiguous conserved sequence of 4 to 5 residues, which is atleast required for the designing of degenerate primers. Surprisingly,the amino acid sequences were more conserved between the Δ5 desaturaseand Δ6 desaturase of the same species than between Δ5 desaturasesobtained from different species. This suggests the presence of aspecies-specific conserved sequence in the cytochrome b5-domain fusiondesaturase gene. To investigate on this, the nucleotide sequences of theMpDES6 and the MpDES of an unknown function, described in Genetics 159,p981, 2001, were compared. As a result, it was found that two amino acidsequences: (I(E/N)(G/D)KVYDV (SEQ ID NO: 41) and DPDI(Q/D)(Y/T)(M/V)P(SEQ ID NO: 42)) were conserved. Sequences of degenerate primerscorresponding to the respective amino acid sequences are as follows:

dΔ5DES-F: (SEQ ID NO: 15) 5′-AT(A/T/C)(A/G)AIG(A/G)IAA(A/G)TITA(T/C)GA(T/C)GT-3′; and dΔ5DES-R: (SEQ ID NO: 16)5′-GGIA(T/C)I(G/T)(A/T)IT(G/C)(A/G/T)AT(A/G)TCIGG (A/G)TC-3′.

PCR was carried out using these primers (dΔ5DES-F and dΔ5DES-R), and theresulting DNA fragments were subcloned. Nucleotide sequences of theclones were determined, and a full-length cDNA was obtained for clonesthat had a target cDNA sequence, using the following primers:

MpDES5-02R: (SEQ ID NO: 17) 5′-GTGTGTACGATCCGTGGTTACC-3′; andMpDES5-01F: (SEQ ID NO: 18) 5′-AAGGCGGGACAGGATTCAACAC-3′.Note that, the materials and methods for the experiment were the same asfor Example 1.

From the cDNA, clones c1 and c2 of different lengths (c1: 2,427 bp; c2:2,285 bp) were isolated as candidates for the Marchantiapolymorpha-derived Δ5 desaturase. By comparing the nucleotide sequencesof the clones c1 and c2, it was found that alternative splicing hadoccurred in a 5′ non-coding region. The alternative splicing did notchange the reading frame, and both clones c1 and c2 coded for 484 aminoacids (SEQ ID NO: 6). The clone c1 of 2,427 bp was used as a MpDES5 gene(SEQ ID NO: 5) in the following Examples.

A comparison of an estimated amino acid sequence of MpDES5 cDNA with anamino acid sequence of the Δ5 desaturase of a filamentous fungus (M.alpina) found 31.4% identity. No comparison was made for the Δ5desaturase of Physcomitrella patens, which is closely related toMarchantia polymorpha, because no sequence information is available forthe Δ5 desaturase of this particular organism.

Example 4 Functional Analysis Using Methylotrophic Yeast (Pichicapastoris)

To examine functions of the respective cDNAs of the MpDES6, MpELO1, andMpDES5, a construct in which an ORF was placed downstream of amethanol-inducible promoter AOX1 was prepared for each gene. Theconstructs were introduced into methylotrophic yeast (Pichia pastoris)to analyze their fatty acid compositions. The ORFs of cDNA nucleotidesequences of the MpDES6, MpELO1, and MpDES5 were PCR-amplified using thefollowing primers.

(Primers for the amplification of MpDES6 ORF) MpD6-17F: (SEQ ID NO: 19)5′-GGAATTCGCGATGGCCTCGTCCACCACCAC-3′; and MpD6-18F: (SEQ ID NO: 20)5′-GGAATTCTACTTTCGCAGCGTATGCTACC-3′. (Primers for the amplification ofMpELO1ORF) MpD6ELO1-15F: (SEQ ID NO: 21)5′-GGAATTCGCGATGGAGGCGTACGAGATGG-3′; and MpD6ELO1-16F: (SEQ ID NO: 22)5′-GGAATTCTTCTGCCTTTTTGCTCTTGATC-3′. (Primers for the amplification ofMpDES5 ORF) MpD5-11F: (SEQ ID NO: 23)5′-GTTGAATTCGACAGTTATGCCGCCACACGC-3′; and MpD5-12R: (SEQ ID NO: 24)5′-GTTGAATTCAGGCCCAAAGCATGCTGTCAC-3′.

These primers contained EcoRI recognition sequences (underlined) andwere used in the following cloning process. Further, PCR was carried outusing 0.5 U of Pyrobest DNA polymerase (Takara) with 20 μl of reactionsolution, in accordance with the method recommended by the manufacturer.The reaction condition was as follows: 94° C. for 2 minutes, followed by25 cycles of reaction at 94° C. for 1 minute, 57° C. for 1 minute, and72° C. for 1 minute, and cooling down to 4° C. Each of the resulting ORFfragments was digested with EcoRI, gel purified by the method describedin Example 1, and ligated in the sense direction to an EcoRI sitedownstream of a methanol-inducible promoter 5′AOX1 in a methylotrophicyeast expression vector pPICZA (Marker: zeocin-resistant gene,Invitrogen).

In order to obtain transformants, expression constructs and a pPICZAvector as a control were introduced into a PPY1-line of methylotrophicyeast using a Pichia EasyComp kit (Invitrogen), using a zeocin-resistantgene as a marker. Note that, the methylotrophic yeast can synthesizelinoleic acid and α-linolenic acid, which are substrates of the Δ6desaturase, but cannot synthesize other precursors of arachidonic acidor EPA.

To express the introduced genes in the transformants, the transformantswere first cultured until OD (600 nm) became 0.5 in a minimal mediumcontaining 1.0% glycerol as a sole carbon source. The transformants werethen cultured at 30° C. for three days in a minimal medium containing0.5% methanol as a sole carbon source, until saturation was reached.Here, an EasySelect Pichia Expression Kit (Invitrogen) was usedaccording to the method recommended by the kit. Thereafter, fatty acidcompositions of the respective transformants were measured using a GC-MSand according to a known method (Biosci. Biotechnol. Biochem. 67, p605,2003).

In an MpDES6 gene-expressing transformant, the products of the Δ6desaturase reaction, g-linolenic acid and stearidonic acid, werecontained in 7.4% and 0.7%, respectively, with respect to the totalfatty acids. In a pPICZA vector-introduced yeast used as a control,γ-linolenic acid and stearidonic acid were not detected. Thus, it wasshown that the MpDES6 encoded the Δ6 desaturase.

In an MpELO1 gene-expressing transformant, 14.1% of the total fattyacids was dihomo-γ-linolenic acid when γ-linolenic acid was added. Onthe other hand, 1.5% of the total fatty acids was eicosatetraenoic acidwhen stearidonic acid was added. In a pPICZA vector-introduced yeastused as a control, dihomo-g-linolenic acid or eicosatetraenoic acid wasnot detected. Thus, it was shown that the MpELO1 encoded the Δ6 chainelongase.

In an MpDES5 gene-expressing transformant, 1.1% of the total fatty acidswas arachidonic acid when dihomo-γ-linolenic acid was added. On theother hand, 0.1% of the total fatty acids was eicosapentaenoic acid(EPA) when stearidonic acid was added. In a pPICZA vector-introducedyeast used as a control, arachidonic acid or eicosapentaenoic acid wasnot detected. Thus, it was shown that the MpDES5 encoded the Δ5desaturase.

It was therefore confirmed that Marchantia polymorpha can be used toobtain MpDES6, MpELO1, and MpDES5, which are genes that encode the Δ6desaturase, Δ6 chain elongase, and Δ5 desaturase, respectively.

Example 5 Reconstruction of Marchantia polymorpha Polyunsaturated FattyAcid Biosynthesis System in a Methylotrophic Yeast (P. pastoris)

To co-express the MpDES6, MpELO1, and MpDES5, EcoRI-digested amplifiedORF fragments of MpELO1 and MpDES5 prepared in Example 4 were ligated toa methylotrophic yeast-expression vector pPIC3K (Marker: HIS4 gene,Invitrogen) and a methylotrophic yeast-expression vector PIC6A (Marker:blasticidin-resistant gene, Invitrogen), respectively. In each vector,the ligation was made in the sense direction at the EcoRI sitedownstream of a 5′AOX1 promoter. For the MpDES6, the expression vectorprepared in Example 4 was used. Hereinafter, the expression vectors forthe MpDES6, MpELO1, and MpDES5 are referred to as pPICZA-MpDES6 vector,pPIC3K-MpELO1 vector, and pPIC6A-MpDES5 vector, respectively.

The pPICZA-MpDES6 vector was transferred into a methylotrophic yeastPPY12 line (his4, arg4) having the same fatty acid composition as themethylotrophic yeast PPY1 used in Example 4. As a control, a pPICZAvector was also transferred. Transformants were obtained using azeocin-resistance marker. Then, the pPIC3K-MpELO1 vector was introducedinto the transformant that has incorporated the pPICZA-MpDES6 in itsgenome and the transformant that has incorporated the pPICZA in itsgenome. The pPIC3K vector was also introduced as a control into thetransformants in the same manner. Transformants were obtained using thehistidine synthesizing ability as a marker. Finally, the pPIC6A-MpDES5vector was introduced into the transformant that has incorporated thepPICZA-MpDES6 and pPIC3K-MpEL01 in its genome, and to the transformantthat has incorporated the pPICZA and pPIC3K in its genome. The pPIC6Avector was also introduced as a control into the transformants in thesame manner. Transformants were obtained using the blasticidinresistance as a marker.

Using the transformants that have incorporated two or three kinds ofgenes, an experiment was conducted to reconstruct the arachidonicacid/EPA biosynthesis system of Marchantia polymorpha. First, using thetransformants that have incorporated the two types of genes (MpDES6 andMpELO1), an MpDES6 protein and an MpELO1 protein were co-expressed inthe methylotrophic yeasts. As a result, g-linolenic acid and stearidonicacid, which are the products of Δ6 desaturation, were contained in 2.9%and 0.4%, respectively, with respect to the total fatty acids, whereasdihomo-g-linolenic acid and eicosatetraenoic acid, which are produced bythe chain elongation of the g-linolenic acid and stearidonic acid,respectively, were contained in 2.8% and 0.2%, respectively, withrespect to the total fatty acids. In the controls, these fatty acidswere not detected. In the transformants that have incorporated threetypes of genes (MpDES6, MpELO1, and MpDES5), production of arachidonicacid (0.1% in the total fatty acids) and eicosapentaenoic acid (EPA,0.03% in the total fatty acids) was confirmed, in addition to theg-linolenic acid, stearidonic acid, dihomo-γ-linolenic acid, andeicosatetraenoic acid, which were contained in 2.8%, 0.5%, 1.5%, and0.1%, respectively, with respect to the total fatty acids. In thecontrols, these fatty acids were not detected. This result showed thatreconstruction of polyunsaturated fatty acid biosynthesis system isindeed possible in organisms other than Marchantia polymorpha, byexpressing Marchantia polymorpha-derived Δ6 desaturase gene, Δ6 chainelongase gene, and Δ5 desaturase gene therein.

Example 6 Construction of a Vector for Rice, and Transfer of the VectorInto Rice

To express the MpDES6 gene, MpELO1 gene, and MpDES5 gene in rice,expression constructs were prepared in the following steps (i) to (iv).FIG. 1 shows the procedure.

In PCR using primers that were designed between a cauliflower mosaicvirus (CaMV) 35S promoter and a NOS terminator of pBI221 (TOYOBO), anexpression vector p35S—NOS not including a β-Glucuronidase (GUS) geneportion was prepared.

Namely, the following primers were used in PCR:

MK001(F): (SEQ ID NO: 25) 5′-CGGGATCCTCTCCTGGCGCACCATCGTC-3′; andMK002(R): (SEQ ID NO: 26) 5′-GGGGTACCAACGCGCTTTCCCACCAACG-3′.

Note that, the primer MK001(F) contained a BamHI recognition sequence(underlined) and was annealed to the 3′ end of the GUS gene. The primerMK002(R) was annealed to the 5′ end of the GUS gene. (BamHI site isupstream of the annealed site.) The PCR was carried out with 50 μl ofreaction solution, using 0.5 U of Pyrobest DNA polymerase (Takara) bythe method recommended by the manufacturer. The reaction conditions wereas follows: 96° C. for 5 minutes, followed by 30 cycles of reaction at94° C. for 30 seconds and 68° C. for 4 minutes, and cooling down to 4°C. The resulting ORF fragments were digested with BamHI, gel purified bythe method described in Example 1, and were self-ligated.

(ii) Next, the ORFs of the MpDES6 gene, MpELO1 gene, and MpDES5 genewere ligated to the XbaI site of p35S—NOS. For the amplification ofORFs, the following primers containing an XbaI recognition sequence(underlined) were used.

(Primers for the amplification of MpDES6 ORF): MpD6-21F: (SEQ ID NO: 27)5′-GCTCTAGAGCGATGGCCTCGTCCACCACC-3′; and MpD6-11R: (SEQ ID NO: 28)5′-GCTCTAGACTATACTTTCGCAGCGTATGC-3′. (Primers for the amplification ofMpELO1 ORF): MpD6ELO1-18F: (SEQ ID NO: 29)5′-GCTCTAGAGCGATGGAGGCGTACGAGATGG-3′; and MpD6ELO1-13R: (SEQ ID NO: 30)5′-GCTCTAGATTATTCTGCCTTTTTGCTC-3′. (Primers for the amplification ofMpDES5 ORF): MpD5 22F: (SEQ ID NO: 31)5′-GCTCTAGAGACAGTTATGCCGCCACACGC-3′; and MpD5 23R: (SEQ ID NO: 32)5′-GCTCTAGAAGGCCCAAAGCATGCTGTCAC-3′.

The PCR was carried out with 20 μl of reaction solution, using 0.5 U ofPyrobest DNA polymerase (Takara) by the method recommended by themanufacturer. The reaction conditions were as follows: 94° C. for 2minutes, followed by 25 cycles of reaction at 94° C. for 1 minute, 57°C. for 1 minute, and 72° C. for 1 minute, and cooling down to 4° C. Theresulting ORF fragments were digested with XbaI, gel-purified by themethod described in Example 1, and were used for cloning.

(iii) By taking advantage of the fact that all of the resultinggene-expression constructs (respectively represented by p35S-MpDES6,p35S-MpELO1, and p35S-MpDES5) had a PstI site at the 5′ end of theCaMV35S promoter and an EcoRI site at the 3′ end of the NOS terminator,expression cassettes for these three genes were ligated to one another.First, PCR was carried out using the primers below, and a p35S-MpDES5 asa template, so as to amplify an expression cassette portion of theMpDES5 gene. The amplified fragment was then cloned into the PstI siteat the 5′ end of the CaMV35S promoter of the p35S-MpDES (see FIG. 1).

(Primers for the amplification of MpDES5-gene expression cassette) M13R:(SEQ ID NO: 33) 5′-CAGGAAACAGCTATGACC-3′; and NOS-R4-PST: (SEQ ID NO:34) 5′-AAACTGCAGATTCCCGATCTAGTAACATAG-3′.

Note that, the M13R primer was annealed to a vector sequence upstream ofthe CaMV35S promoter. Further, the NOS-R4-PST primer contained a PstIrecognition sequence (underlined) and was annealed to the 3′ end of theNOS terminator. The EcoRI site at the 3′ end of the NOS terminator wasnot contained.

The PCR was carried out with 20 μl of reaction solution, using 0.5 U ofPyrobest DNA polymerase (Takara) by the method recommended by themanufacturer. The reaction conditions were as follows: 94° C. for 2minutes, followed by 25 cycles of reaction at 94° C. for 1 minute, 57°C. for 1 minute, and 72° C. for 1 minute, and cooling down to 4° C. Theresulting DNA fragments were digested with PstI, gel-purified by themethod described in Example 1, and cloned into the PstI site of theplasmid (p35S-MpDES6) containing the MpDES6-gene expression cassette.

(iv) To the resulting construct in which the expression cassettes of theMpDES5 gene and MpDES6 gene were ligated (represented by“p35S-MpDES5/35S-MpDES6”), an expression cassette of the MpELO1 gene wasfurther ligated. PCR was carried out using the primers below, and ap35S-MpELO1 as a template, so as to amplify an expression cassetteportion of the MpELO1 gene. The amplified fragment was then cloned intothe EcoRI site at the 3′ end of the NOS terminator in the MpDES6-geneexpression cassette.

(Primers for the amplification of MpELO1-gene expression cassette)35S-F3-EI: (SEQ ID NO: 35) 5′-CCGGAATTCGCATGCCTGCAGGTCCCCAGA-3′; andM13F: (SEQ ID NO: 36) 5′-TGTAAAACGACGGCCAGT-3′.

Note that, the 35S—F3-EI primer contained an EcoRI recognition sequence(underlined) and was annealed to the 5′ end of the CaMV35S promoter.Further, the M13F primer was annealed to a vector sequence downstream ofthe NOS terminator.

The PCR was carried out with 20 μl of reaction solution, using 0.5 U ofPyrobest DNA polymerase (Takara) by the method recommended by themanufacturer. The reaction conditions were as follows: 94° C. for 2minutes, followed by 25 cycles of reaction at 94° C. for 1 minute, 57°C. for 1 minute, and 72° C. for 1 minute, and cooling down to 4° C. Theresulting DNA fragments were digested with EcoRI, gel-purified by themethod described in Example 1, and cloned into the EcoRI site of theconstruct (p35S-MpDES5/35S-MpDES6) in which the MpDES5-gene expressioncassette and the MpDES6-gene expression cassette were ligated.

By these procedure, an expression construct(p35S-MpDES5/35S-MpDES6/p35S-MpELO1) was prepared in which the threegenes-expression cassettes of MpDES5, MpDES6, and MpELO1 were ligated inthis order.

The construct so obtained was introduced into rice, together with aplasmid having bialaphos as a selection marker, using a particle gun bya known method (Genes Genet. Syst. 73, p219, 1998). As a result, atransformed rice was obtained.

Example 7 Reconstruction of Marchantia polymorpha Polyunsaturated FattyAcid Synthesis System in Tobacco (N. tabacum SR-1)

This Example confirmed that the foregoing Marchantia polymorpha-derivedunsaturated fatty acid synthetase genes, i.e. the MpDES6 gene, MpDES5gene, and MpELO gene were indeed well functional in plants.

More specifically, by introducing the MpDES6 gene, MpDES5 gene, andMpELO gene into tobacco, production of arachidonic acid and other fattyacids were confirmed. For comparison, a tobacco was prepared into whichfilamentous fungus (M. alpina)-derived Δ6 desaturase gene (MaDES6), Δ5desaturase gene (MaDES5), and Δ6 fatty-acid-chain elongase (MaELO) wereintroduced.

(i) Construction of a Vector (pSPB1519) Containing FilamentousFungus-Derived Genes

The pE2113 (Mitsuhara et al. Plant Cell Physiol. 37, 45-59 1996) has acauliflower mosaic virus 35S (E1235S) promoter, having repeatingenhancer sequences, and a nopaline synthase (nos) terminator.

The pE2113 was digested with SnaBI and then ligated to an XhoI linker(TAKARA) to obtain a plasmid. The resulting plasmid was digested withSacI, blunted, and ligated to a BamHI linker (TAKARA) to obtain pUE7. Ofthe DNA fragments obtained by the digestion of pUE7 with HindIII andEcoRI, a fragment having an E1235S promoter was ligated to aplant-transformation binary vector pBINPLUS (van Engelen et al.Transgenic research 4, p288, 1995) digested with HindIII and EcoRI. As aresult, pSPB505 was obtained.

Meanwhile, a plasmid pMLD101 containing the MaDES6 gene was digestedwith XhoI followed by partial digestion with BamHI, so as to obtain anabout 1.6 kb DNA fragment. The DNA fragment so obtained was ligated to aDNA fragment of a binary vector obtained by the digestion of pSPB505with XhoI and BamHI. As a result, pSPB559 was obtained.

The pUCAP (van Engelen et al. Transgenic research 4, p288, 1995) wasdigested with AscI, blunted, and ligated to a PacI linker to obtainpUCAPP.

The pE2113 was digested with SnaBI and ligated to a BamHI linker(TAKARA) to obtain pUE6. This pUE6 was digested with SacI, blunted, andligated to a SalI linker (TAKARA) to obtain pUE8. Of the DNA fragmentsobtained by the digestion of pUE8 with HindIII and EcoRI, a fragmenthaving an E1235S promoter was inserted into the HindIII-EcoRI site ofpUCAPP. A DNA fragment obtained by the digestion of this plasmid withBamHI and SalI was ligated to a DNA fragment obtained by the digestionof cDNA of the MaELO gene with BamHI and XhoI, so as to obtain pSPB1130.The pSPB1130 was digested with PacI, and the resulting DNA fragment ofabout 2.3 kb was inserted into a PacI site of pBINPLUS. A plasmid havingthe same transcription direction for the MaELO gene and the nptII geneon the pBINPLUS were selected as pSPB1157P.

Further, the pSPB599 was digested with PacI, blunted, and an AscI linkerwas inserted to prepare pSPB599A. The pSPB599A was digested with AscI,and a DNA fragment containing the MaDES6 gene, obtained from thedigestion of pSPB599A with AscI, was inserted into the AscI site ofpSPB1157P to obtain pSPB1157.

An about 1.3 kb DNA fragment obtained from the digestion of pCGP1364(Plant Cell Physiol. 36, p1023, 1995) with HindIII and SacII was ligatedto an about 2.9 kb DNA fragment obtained by digesting pCGP1364 withPstI, blunting it, and digesting it with SacII. These DNA fragments werefurther ligated to an about 2.7 kb DNA fragment obtained by digestingpUCAPA with SacI, blunting it, and digesting it with HindIII. As aresult, pSPB184 was obtained.

Meanwhile, from a pCRII vector into which the MaDES5 gene was subcloned,a DNA fragment containing the MaDES5 gene was excised by digesting withXbaI and KpnI. The resulting DNA fragment was ligated to a DNA fragmentobtained by the digestion of the pSPB184 with XbaI and KpnI, so as toobtain pSPB1519A. A DNA fragment obtained by digestion of the pSPB1519Awith AscI was inserted into the AscI site of pSPB1157 to obtainpSPB1519. The pSPB1519 was digested with AscI and inserted into the AscIsite of pSPB1157 to obtain pSPB1519. The MaDES6 gene, MaDES5 gene, andMaELO gene were transcribed in the same direction on the plasmidpSPB1519, and were controlled by the same constitutive promoter.

(ii) Construction of Marchantia polymorpha-Derived Gene Vector(pSPB2368)

The pUCAP (van Engelen et al. Transgenic Research 4, 288-290, 1995) wasdigested with AscI, ligated to a SgfI linker. By further digesting itwith PacI followed by ligation to a FseI linker, pUCSAPF was obtained.In the same manner, InpBINPLUS was processed to obtain pBINSAPF.

In addition, the pUC19 was digested with HindIII and ligated to a PacIlinker. By further digesting it with EcoRI followed by ligation to aFseI linker, pUCPF was obtained as a subcloning vector. Further, thepUC19 was digested with HindIII and ligated to a SgfI linker. By furtherdigesting it with EcoRI followed by ligation to an AscI linker, pUCSAwas obtained. A vector in which E1235S was inserted into theHindIII-XbaI site of pUCSAPF, and in which a mannopin synthetase (mas)gene terminator was inserted into the SacI-EcoRI site of pUCSAPF wasdigested with XbaI and SacI and blunted to obtain pSPB2353A. To a bluntend of pSPB2353A, a DNA fragment containing the MaDES6 gene which wasexcised from the p35S-MpDES6 with XbaI and blunted was ligated. As aresult, pSPB2353 was obtained.

A vector in which E1235S was inserted into the HindIII-XbaI site ofpUCSA, and in which a mannopin synthetase (mas) gene terminator wasinserted into the SacI-EcoRI of pUCSA was digested with XbaI and SacI toobtain pSPB2355A.

Meanwhile, using the p35S-MpELO1 as a template, PCR was carried outusing the following primers:

XbaMpELOf: (SEQ ID NO: 43) 5′-AGTCTCTAGAGCGATGGAGGCGTACG-3′; andSacMpELOr: (SEQ ID NO: 44) 5′-CAGTGAGCTCGGTGTCTTATTCTGCC-3′.

PCR was run using a highly accurate KOD-plus-DNA polymerase (Toyobo) asan enzyme. The reaction was carried out at a maintained temperature of94° C. for two minutes, followed by 25 cycles of reaction at 94° C. for15 seconds and at 68° C. for 1 to 3 minutes. An MpELO DNA fragment soprepared was digested with XbaI and SacI, and was ligated to thepSPB2355A to obtain pSPB2355. Further, a DNA fragment obtained by thedigestion of pSPB2355 with SgfI and AscI was ligated to pSPB2353digested with SgfI and AscI. As a result, pSPB2361 was obtained.

A vector in which E1235S was inserted into the HindIII-XbaI site ofpUCPF, and in which a mannopin synthetase (mas) gene terminator wasinserted into the SacI-EcoRI site of pUCPF was digested with XbaI andSacI to obtain pSPB2352A.

Meanwhile, using the p35S-MpDES5 as a template, PCR was carried outusing the following primers:

XbaMpD5f: (SEQ ID NO: 45) 5′-AGCTTCTAGAGCCATGCCGCCACACGCCC-3′; andSacMpD5r: (SEQ ID NO: 46) 5′-CAGTGAGCTCTCAGCCATCCAGTCGT-3′.The PCR conditions were the same.

An MpD5 DNA fragment prepared by the PCR was digested with XbaI and SacIand ligated to pSPB2352A to obtain pSPB2352.

A DNA fragment obtained by the digestion of pBINSAPF with PacI and FseIwas ligated to a DNA fragment containing the MpDES5 gene which wasexcised from pSPB2352 with PacI and FseI. As a result, pSPB2368A wasobtained. Further, pSPB2368A was digested with SgfI and PacI and ligatedto a DNA fragment containing the MpDES6 and MpELO genes which wereexcised from pSPB2361 with SgfI and PacI. As a result, pSPB2368 wasobtained. The MpDES6 gene, MpDES5 gene, and MpELO gene were transcribedin the same direction on the plasmid pSPB2368, and were controlled bythe same constitutive promoter.

(iii) Gene Introduction into Tobacco

Next, according to a known method (Plant J. 5, 81, 1994), Agrobacteriumtumefaciens (strain: Ag10 (Lazo et al. 1991, Biotechnology 9: 963-967))was transformed using pSPB2368 or pSPB1519. The transformedAgrobacterium having pSPB2368 or pSPB1519 was used to infect a tobaccoleaf disk. From the transgenic tobacco leaf so obtained, RNA wasextracted using a RNeasy Plant Mini Kit (Qiagen), and a line expressingthe introduced gene was selected by RT-PCR using an ordinary method.

From the tobacco leaf that has incorporated the pSPB1519 containing thefilamentous fungus-derived enzyme gene (pSPB1519-transformed tobacco),and from the tobacco leaf that has incorporated the pSPB2368 containingthe Marchantia polymorpha-derived enzyme gene (pSPB2368-transformedtobacco), lipids were extracted according to known methods (YasuhikoFUJINO, “Seibutsu-Kagaku Jikken-ho (Method of Biochemical Experiment)9”, Gakkai Shuppan Center (1978); Akihiro YAMADA, “Seibutsu-KagakuJikken-ho (Method of Biochemical Experiment) 24”, Gakkai Shuppan Center(1989)). The lipids were analyzed by gas chromatography (HewlettPackard, HP-6800). The result of analysis is shown in Table 1.

Note that, the same analysis was carried out using, as a control, atobacco leaf into which no gene was introduced.

TABLE 1 Control Control pSPB2368 pSPB2368 pSPB1519 (%) (mg/gFW) (%)(mg/gFW) (%) Linoleic acid 9.55 1.17 1.37 0.2 9.51 α-linolenic acid49.99 6.12 17.83 2.58 39.24 γ-linolenic acid 0 0 4.06 0.59 3.37Dihomo-γ-linolenic 0 0 10.85 1.57 3.09 acid Arachidonic acid 0 0 10.271.49 0 Eicosatetraenoic 0 0 4.89 0.71 0 acid Eicosapentaenoic 0 0 2.680.39 0 acid Total amount of — 12.25 — 14.48 — lipids

In this Example, gas chromatography analysis was made under thefollowing conditions:

(Gas Chromatography Analysis Condition)

Column: Supelco SP-2330, Fused Silica Capillary Column, 30 m×0.32 mm ID,0.2 μm;

Temperature: Inj: 240° C., Det: 250° C., Oven: 180° C. for 3 min, 180°C.→220° C. (2° C./min); and

Column flow rate: 30 cm/sec, Pressure: 200 kPa, Detector: FID.

Each peak in the chromatogram was determined by a retention time ofmethyl ester of normal fatty acids and GC-MASS (Hewlett Packard,HP-5973) analysis, and the proportions of the respective fatty acidswere determined in accordance with a peak area. In Table 1, “Control”indicates a control, “pSPB2368” indicates the pSPB2368-transformedtobacco, “pSPB1519” indicates the pSPB1519-transformed tobacco.

The results shown in Table 1 confirmed accumulation ofdihomo-γ-linolenic acid in the tobacco that has incorporated thepSPB1519 containing the filamentous fungus-derived genes(pSPB1519-transformed tobacco), but no accumulation of arachidonic acid.On the other hand, in the tobacco that has incorporated the pSPB2368containing the Marchantia polymorpha-derived enzyme genes(pSPB2368-transformed tobacco), accumulation of not only arachidonicacid but also eicosatetraenoic acid and eicosapentaenoic acid wasconfirmed. These results suggest that, in higher plants, the Marchantiapolymorpha-derived enzymes are functionally superior to the filamentousfungus-derived enzymes in the ability to synthesize arachidonic acid andother polyunsaturated fatty acids using linoleic acid and α-linolenicacid as substrates.

Abbadi has reported that by introducing Phaeodactylumtricornutum-derived Δ6 desaturase and Δ5 desaturase, and aPhyscomitrella patens chain elongase gene into tobacco and flax (Linumusitatissimum), 1.5% arachidonic acid was accumulated in a seed oftobacco, and 1.0% arachidonic acid was accumulated in a flax (AmineAbbadi et al. Plant Cell 16, 2734-2748, 2004).

In the present Example, by introducing Marchantia polymorpha-derived Δ6desaturase, Δ5 desaturase, and chain elongase into tobacco, 10% orgreater arachidonic acid was accumulated in a tobacco leaf. This resultsuggests that the pSPB2368-transformed tobacco in the present Example iscapable of more efficiently synthesizing polyunsaturated fatty acids, ascompared with the foregoing report.

Further, it has been reported that as a result of lipid modification ofArabidopsis thaliana using three types of genes: Isochrysisgalbana-derived Δ9 chain elongase, Englena gracilis-derived Δ8desaturase, and filamentous fungus-derived Δ5 desaturase, 6.6 mol % ofarachidonic acid and 22.5 mol % of fatty acids having 20 or more carbonatoms with respect to the total lipids were contained in the leaf(Baoxiu Qi et al. Nature Biotechnology 22, 739-745, 2004). In thisreport, polyunsaturated fatty acids are synthesized through amodification pathway different from the pathway using the Δ6 desaturase,Δ5 desaturase, and chain elongase, and it was found that morepolyunsaturated fatty acids could be accumulated when Marchantiapolymorpha-derived enzymes were used, although a simple comparisoncannot be made.

The pSPB2368-transformed tobacco having a 30% or greater modified fattyacid content with respect to the total lipids showed no morphologicalabnormalities. Further, since the pSPB2368-transformed tobacco has noproblem in terms of fertility and bore a lot of seeds, it is believedthat the ectopical increase of the polyunsaturated fatty acids has nosignificant effect on the growth and development of plants.

In the transgenic plants reported so far, up to about 20% of totallipids is polyunsaturated fatty acids having 20 or more carbon atoms.The value reduces to about 6% when only arachidonic acid is considered.However, as in the present Example, the use of Marchantiapolymorpha-derived fatty acid synthetases makes it possible to go beyondthis limitation and produce more polyunsaturated fatty acids in plants.

Specific embodiments or examples implemented in the best mode forcarrying out the invention only show technical features of the presentinvention and are not intended to limit the scope of the invention.Variations can be effected within the spirit of the present inventionand the scope of the following claims.

INDUSTRIAL APPLICABILITY

The present invention provides a Δ5 desaturase gene, a Δ6 desaturasegene, and a Δ6 chain elongase gene, which are obtained from a singlespecies of Marchantiales. The three genes, when expressed together in aplant, function more desirably in the plant as compared with the casewhere genes obtained from organisms of different species are expressed.Further, since Marchantiales can be considered as a model plant ofhigher plants, the genes derived from Marchantiales can function moredesirably in plants than genes derived from non-plants.

Further, with a transformant according to the present invention,polyunsaturated fatty acids such as arachidonic acid or eicosapentaenoicacid (EPA) can be produced. More advantageously, a transformantaccording to the present invention produces these fatty acids at lowcost by an environmentally friendly process. The product arachidonicacid or EPA can be inexpensively marketed as a material with differentpurposes. When used as food, the transformant can increase the value ofthe product with its high arachidonic acid or EPA content.

As described above, genes and proteins of the present invention areuseful in producing arachidonic acid and EPA. Further, transformantsthat have incorporated genes of the present invention are highly usefulin the arachidonic acid or EPA production in pharmaceutical industry,food industry, and various other material industries. The usefulness ofthe transformant is particularly notable in agricultures when thetransformant is a plant, because the transformation increasesarachidonic acid and EPA levels in plants.

1-8. (canceled)
 9. An isolated Marchantiales-derived gene thathybridizes under stringent conditions to all of or part of SEQ ID NO: 5or a complementary sequence thereto, wherein said gene encodes a proteinhaving Δ5 fatty acid desaturating activity.
 10. The isolated gene ofclaim 9, wherein said gene (a) consists of a nucleotide sequence of SEQID NO: 5, or (b) hybridizes under stringent conditions to SEQ ID NO: 5or a complementary sequence thereto.
 11. The isolated gene of claim 9,wherein said gene (a) comprises a nucleotide sequence of nucleotides 375to 1829 of SEQ ID NO: 5, or (b) hybridizes under stringent conditions toa nucleotide sequence of nucleotides 375 to 1829 of SEQ ID NO: 5 or acomplementary nucleotide sequence thereto.
 12. An isolated gene encodinga Marchantiales-derived protein having Δ5 fatty acid desaturatingactivity, wherein said protein has an amino acid sequence of SEQ ID NO:6, or wherein said gene encodes a protein with an amino acid sequencethat has been modified by substitution, deletion, insertion, and/oraddition of one or more amino acids of SEQ ID NO:
 6. 13-28. (canceled)29. A recombinant expression vector comprising the isolatedMarchantiales-derived gene of claim
 9. 30. A transformed cell comprisingthe recombinant expression vector of claim
 29. 31. A plant, a progeny ofsaid plant, a vegetatively propagated plant of said plant, or a tissueof said plant, wherein the isolated Marchantiales-derived gene of claim9 is expressed in the plant, the progeny of said plant, the vegetativelypropagated plant of said plant, and the tissue of said plant.
 32. Theplant, the progeny of said plant, the vegetatively propagated plant ofsaid plant, or the tissue of said plant of claim 31, wherein said plant,the progeny of said plant, the vegetatively propagated plant of saidplant, or the tissue of said plant has a modified fatty acidcomposition.
 33. A method of producing fatty acids comprising growingthe plant of claim 31, and obtaining said fatty acids from said plant orplant tissue.
 34. A method of modifying a fatty acid compositioncomprising expressing the isolated nucleic acid of claim 9 in a plant.